Polymerases are commonly stored in a buffer that includes Ethylenediaminetetraacetic acid (EDTA) and a detergent. At the time of use, the enzyme in its storage buffer is diluted usually at least 10 fold when added to a reaction mix containing dNTPs, primers, probes and nucleic acid template for DNA amplification. Typically the storage buffer is designed to optimize storage of the enzyme reagents and not to optimize an amplification reaction. This is because the storage buffer is generally diluted at least 10 fold so that components that might interfere with the reaction are diluted out.
Large numbers of samples may be analyzed by probe quantitative polymerase chain reaction (probe qPCR) or reverse transcription qPCR (RT-qPCR) for clinical nucleic acid-based diagnostics and certain research projects. Examples include gene expression profiling or determining thresholds of biological material in environmental samples. An advantage of probe qPCR master mixes is that only a single dilution step is required when the master mix is added to the nucleic acid template to provide optimal reaction conditions for probe qPCR.
Master mixes differ from storage buffers containing enzyme reagents and high concentrations of glycerol for reasons that include the presence in the master mix of reagents that are common to reactions including dNTPs, magnesium ions, and in some cases, probes and primers, but never sample nucleic acid templates which are unique to each reaction. Moreover, because master mixes are typically diluted only 2 fold when added to the nucleic acid template regardless of whether the nucleic acid is in a biological fluid or a formulated buffer, all the components in the master mix should be compatible with optimal reaction conditions.
Consequently, it is desirable that a probe PCR master mix be both suitable for storage and compatible with reaction conditions for probe qPCR. The master mix should provide consistency of results and not compromise sensitivity of the reaction.
Unfortunately, the consistent activity of stored master mixes for use in probe qPCR and the associated sensitivity of the assay diminishes over time reducing confidence in the data thereby undermining the use of these preparations.